Effects of age and sex on protein abundance in the mouse heart

ABSTRACT: We compared protein abundance in the left and right ventricles of male and female C57BL6/J mice at 4 months and 20 months old. Frozen tissues were weighed, thawed, and cut into small pieces (2-3 mm3) on weigh boats on ice. Tissue pieces were transferred to 2.0 mL eppendorf tubes. Cold RIPA (Thermo #89901) was added to tissues at a ratio of 20:1 (20 µL RIPA per 1 mg of tissue) and samples were homogenized as above then sonicated with a handheld sonicator (Fisher #FB120110) at 40% amplitude for 15 cycles (each cycle is 1 s of sonication, followed by 5 s pause; three rounds. The samples were centrifuged at 14,000 × g for 15 min at 4°C, and the supernatants were collected. Soluble protein concentration was measured with the BCA protein assay kit following manufacturer instruction; 100 µg of proteins were digested using a filter-assisted protocol as described (Manza et al., 2005) with a 3-hr LysC digestion and 12-hr trypsin digestion. Peptide digest were tagged with tandem mass tags (TMT; 10-plex reagent, Thermo #90110) following manufacturer’s instructions. Tagged digests within the same block are then mixed and fractionated with the high-pH reversed phase peptide fractionation kit (Thermo #84868). Fractionated peptides are dried and resuspended in 0.1% formic acid (LC-MS grade, thermo #28905) at a final concentration of 0.5 µg/µL. Each peptide fraction (~1.5 µg) was further separated with online low-pH reversed-phase LC (PepMap C18 column, 3-μm particle, 100-Å pore; 75 μm x 150 mm; Thermo Fisher Scientific) via the EASYnLC 1200 system coupled to the Easy-Spray ion source (Thermo Fisher Scientific) at 300 nL/min with a 120 min gradient: 0 -105 min: 0 to 40% B; 105 - 110 min: 40 to 70% B; 110 - 115 min: 70 to 100% B; 115 - 120 min: 100% B (solvent A: 0.1% v/v formic acid; solvent B: 80% v/v acetonitrile; column temperature: 50 °C). Mass spectra were acquired on a Thermo Scientific Q-Exactive HF Orbitrap mass spectrometer with the following settings: polarity: positive; data dependent acquisition (DDA): top 15 ions, MS resolution: 60,000, mass range: 300-1650 m/z; precursor dynamic exclusion: 30s, maximum ion injection time: 20 ms; MS automatic gain control (AGC) target: 3e6. isolation window: 1.4 m/z; stepped normalized collision energy (NCE): 28, 30, 32. MS2 resolution; 60,000; MS2 maximum ion injection time: 100 ms; MS2 AGC target: 2e5.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Maggie Lam 

PROVIDER: PXD033719 | JPOST Repository | Tue Nov 08 00:00:00 GMT 2022

REPOSITORIES: jPOST